Along with surgery and radiotherapy, chemotherapy continues to be an effective therapy for many cancers. In fact, several types of cancer are now considered to be curable by chemotherapy and include Hodgkin's disease, large cell lymphoma, acute lymphocytic leukemia, testicular cancer and early stage breast cancer. Other cancers such as ovarian cancer, small cell lung and advanced breast cancer, while not yet curable, are exhibiting positive response to combination chemotherapy.
One of the most important unsolved problems in cancer treatment is drug resistance. Drug resistance includes both intrinsic resistance at the time of treatment using chemotherapy and acquired drug resistance. This problem is a reason for the added importance of combination chemotherapy, as the therapy both has to avoid the emergence of resistant cells and to kill pre-existing cells which are already drug resistant.
Anthracyclines represent an important class of oncolytic agents. Doxorubicin, an anthracycline, which is also known in the art as Adriamycin.TM., is a drug of choice in the clinical management of breast cancer. Therapy with anthracyclines such as doxorubicin is complicated by the appearance of the anthracycline resistant phenotype which limits or negates the oncolytic activity of doxorubicin.
Topoisomerase inhibitors represent a further class of oncolytic agents. Epipodophyllotoxins such as Etoposide.RTM. and Teniposide.RTM. are topoisomerase inhibitors which are useful in the therapy of neoplasms of the testis, small-cell lung and other lung, breast, Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia and Karposi's sarcoma. The therapeutic utility of the epipodophyllotoxins is limited by the appearance of the epipodophyllotoxin resistant phenotype.
One form of multi-drug resistance (MDR) is mediated by a 170-180 kD energy-dependent efflux pump designated as P-glycoprotein, P-gp. P-gp has been shown to play a major role in the intrinsic and acquired resistance of a number of human tumors against hydrophobic, natural product drugs. Drugs that act as substrates for and are consequently detoxified by P-gp include the vinca alkaloids (vincristine and vinblastine), anthracyclines (Adriamycin), and epipodophyllotoxins (etoposide). While P-gp-associated MDR is a major determinant in tumor cell resistance to chemotherapeutic agents, it is clear that the phenomenon of MDR is multifactorial and involves a number of different mechanisms. One such alternative pathway for resistance to anthracyclines involves the emergence of a 190 kD protein that is not P-gp. See McGrath, T., Latoud, C., Arnold, S. T., Safa, A. R., Felsted, R. S., and Center, M. S. Biochem. Pharmacol., 38: 3611, (1989). P190, also referred to as MRP, is found on the plasma membrane and also appears to be localized in the endoplasmic reticulum See Marquardt, D. and Center, M. S., Cancer Res., 52: 3157, (1992).
MRP possesses a nucleotide binding domain that is homologous with the ATP binding site of P-gp. See Marquardt, D., McCrone, S., and Center M. S., Cancer Res., 50: 1426, (1990). The mechanism(s) utilized by P190 to confer resistance to Adriamycin is not well understood but may involve the intracellular redistribution of Adriamycin away from the nucleus. See Marquardt, D. and Center, M. S., supra. Adriamycin is an inhibitor of topoisomerase II (Beck, W. T., Bull. Cancer, 77: 1131, (1990), which is an enzyme involved in DNA replication. Redistribution of Adriamycin away from the nucleus would therefore be an important component in cellular resistance to this drug. The studies published to date on P190 have utilized cell lines selected in vitro for resistance to Adrianycin (McGrath, T., Latoud, C., Arnold, S. T., Safa, A. R., Felsted, R. S., and Center, M. S., supra; Marquardt, D. and Center, M. S., supra; and Marquardt, D., McCrone, S., and Center M. S. Cancer Res., supra. The association of MRP (P190) with drug resistance was made by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of radioactive extracts prepared from Adriamycin-resistant HL60/Adr human leukemia cells labeled with 8-azido-alpha[.sup.32 P]ATP. See McGrath, T., Latoud, C., Arnold, S. T., Safa, A. R., Felsted, R. S., and Center, M. S., supra. The drug-resistance phenotype conferred by P190 is not limited to the anthracyclines. Epipodophyllotoxin resistance is linked to P190 expression. The IC.sub.50 s of HL60/S cells treated with Adriamycin and Etoposide were 0.011 .mu.g/ml and 0.39 .mu.g/ml respectively. The IC.sub.50 S for HL60/Adr cells (a HL60-derived cell line which is resistant to doxorubicin) treated with Adriamycin and Etoposide were 2.2 .mu.g/ml and &gt;10 .mu.g/ml respectively. HL60/S and HL60/Adr cell lines do not express P-glycoprotein. HL60/Adr expresses P190. Thus, resistance to the anthracyclines and epipodophyllotoxins results from P190 expression.
The present invention embraces the elucidation of the role which MRP plays in multiple drug resistance. MRP functions comprise an energy dependent transporter function to transport a range of oncolytics which include compounds which are generally conjugates of lipophilic compounds having anionic groups such as cysteinyl, carboxyl, cysteinylglycine, glutatione S, glucuronic acid or sulfate groups.
Biochemical characterization of the MRP protein allowed the development of MRP-based assays for compounds which reverse MDR by blocking the transport function of MRP, which in turn led to the identification of the compounds of the present invention.